Identification And Activity Assay Of The SezAT Toxin-antitoxin System Of Highly Pathogenic Streptococcus Suis Serotype2

Streptococcus suis serotype2(S. suis2) has been regarded as an important zoonoticpathogen in the world and is responsible for a variety of life-threatening infections inhumans and pigs, including arthritis, meningitis, septicaemia, acute death and so on. InDenmark the first human infection case of S. suis2was found and reported, then it has beenreported all over the world. It was previously thought that S. suis2caused only sporadiccases in humans. However, two recent large-scale outbreaks in human and pigs caused by S.suis2in China in1998and2005have provoked considerable public health concernsworldwide. These two outbreaks showed a prevalent feature of streptococcal toxic shocksyndrome (STSS), with clinical symptoms as acute high fever, shock, acute multiple organfailures. STSS can lead to short course of disease and high lethality, then cause seriouspublic health emergencies.Previously, our joint research group studied intensively in the two highly pathogenicstrain98HAH12and05ZYH33isolated from Chinese STSS patients in Jiangsu andSichuan Provinces. Through whole-genome sequencing and functional annotation, thesequences of these two strains were compared with that of the P1/7reference strain releasedby Sanger center. We identified an unique pathogenicity island (PAI) designated89K whichis specific to the STSS-causing epidemic strains of S. suis2. Further bioinformatics analysisof the89K PAI revealed that it encodes several virulence-related factors, such as theSalK/R two-component signal transduction system (TCS), type IV-like secretion system(T4SS-like), and a zeta toxin. We have provided experimental evidence that the SalK/SalRsystem really involved in the pathogenesis of S. suis2infection, and the T4SS-like systemcontributes to the development of STSS.Recently, we demonstrated that the89K PAI can spontaneously excise from thechromosome to form an extrachromosomal circular product, and the89K excision intermediate acts as a substrate for lateral transfer to non-89K S. suis2recipients. However,attempts to cure this island from wild-type05ZYH33to generate an89K-negative variantby using mitomycin C, an acknowledged plasmid-curing agent, have so far beenunsuccessful. We presume that some key factors may promote the stability of the89K PAI.Further bioimformatics analysis revealed a unique toxin-antitoxin system (TA system)designated SezAT locates in the89K PAI. Among them SezA was antitoxin protein, SezTwas toxin protein. The TA systems were recently identified as a kind of key factors whichmight promote the stability of some mobile genetic elements such as plasmids and genomicislands. Therefore, the highly pathogenic S. suis289K PAI coding SezAT was likely toparticipate in the89K PAI stability of mechanism. But bioinformatics analysis results canonly be hints, whether the TA system has the biological functions still need to beinvestigated. In order to evaluate the activity of the SezAT system and then to furtherevaluate the role of this system in the maintenance of the89K PAI so as to investigate thewhole function of the89K island in S. suis2, specific experiments were conducted andresults included the following aspects:1. Bioinformatics analysis of the SezAT system and RT-PCR assay: Bioinformaticsanalysis revealed that genes05SSU0936(sezT)and05SSU0937(sezA) in the89Kpathogenicity island (PAI) encoded a putative TA system (named SezAT) homologous to theepsilon-zeta TA system from S. pyogenes. These two ORFs have a base of overlap,suggesting co-transcription of these two ORFs. RT-PCR assay confirmed that sezAT were inthe same operon.2. Construction of recombinant expression plasmid and activity analysis of theSezAT system: To construct the recombinant plasmid pJS-AT, the full length of sezA andsezT were amplified and inserted into the double promoter expression plasmid pJS298,respectively.Then the recombinant plasmid pJS-AT was introduced into E. coli BL21(DE3)through the method of electrotransformation. Inducible expression of the SezAT systemwith certain concentration inducers of IPTG (final concentration of1mM) and L-Arabinose(0.2%), respectively. The OD600value of each culture were determinated at different timepoints, and the growth curves were then drawn. The results showed that over-expression ofSezT in E. coli led to severe growth inhibition of the host bacteria, while this toxicity wascounteracted by the expression of SezA. We demonstrated that the SezAT constitutes a active TA system.3. Construction of knockout mutants of the SezAT system: The flanking DNAsequences to sezT or sezAT were amplified from the chromosomal DNA of S. suis05ZYH33and were cloned into a pUC18vector. the SpcRgene cassette amplified from theE. coli-S.suis shuttle vector pSET2was inserted to generate the SezAT knockout vectorspUC::SezT and pUC::SezAT. To obtain the isogenic mutant sezT or sezAT, the competentcells of05ZYH33were subjected to electrotransformation with pUC::SezT or pUC::SezAT,respectively. For all the SpcRtransformants, the suspected mutants were screened andidentified through a preliminary PCR using specific primers for the toxin-encoding genesezT and later multiple PCR with a series of specific primers. We successfully constructedthe isogenic mutant sezT, but failed to get the mutant sezAT. The basic biologicalcharacteristics and pathogenicity of sezT were compared with those of the wild-type strain05ZYH33, no significant differences were observed.In conclusion, a unique toxin-antitoxin system SezAT has been identified from the89KPAI in Chinese isolates of highly pathogenic S. suis205ZYH33. Through thebioinformatics analysis and RT-PCR assay we confirmed that the sezAT system are in thesame operon. Over-expression the SezA or SezT proteins in E. coli BL21(DE3) revealedthat the SezAT system is an active TA system. Our data confirm, for the first time, that the89K PAI encoded an active TA system which is presumed to be requisite for the stability ofthe89K PAI in S. suis2. At the same time we succeed in construcing the mutant sezT. Infuture study great efforts would be made to cure the89K island from the SS2genome basedon the isogenic knockout mutant of SezAT we will use the mutation sezT as thefoundation, through the method of electrotransformation to eliminate the89K island fromthe S. suis2chromosome, aiming to gain the mutation of89K PAI. The results of ourwork will not only expand the development and repertoire of the TA systems, but also behelpful to elucidate the relationship between the89K PAI and the development of STSS.